LARVICIDAL EFFECTS OF CURCUMA LONGA …multidisciplinaryjournal.globalacademicresearchinstitute...Interntional Conference on Health and Medicine 6 Sri Lankan anopheline (Gunathilaka,

Interntional Conference on Health and Medicine

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LARVICIDAL EFFECTS OF CURCUMA LONGA ETHANOL RHIZOME

EXTRACT ON ANOPHELES TESSELLATUS

1Kaushalya Perera , 2 Koshala De Silva

[email protected]

ABSTRACT

Adult Anopheles tessellatus is a vector of Malaria, which is responsible for increased

morbidity and mortality in many tropical and subtropical countries. During epidemics,

the emphasis is given to the use of insecticides as a control measure against mosquitoes.

As an alternative to synthetic insecticides, the use of bio-degradable phytochemicals

against mosquito larvae is considered to be one of the safest approaches to control

mosquito-borne diseases. This project was carried out to monitor the effect of ethanolic

rhizome extract of Curcuma longa on second larval instar of Anopheles tessellatus.

Bio-assay was performed to determine the percentage mortality of Anopheline larvae

following exposure to different concentrations of plant extract. The study indicated that

the mortality rate following exposure increases in a time and dose-dependent manner.

The plant demonstrated its strongest larvicidal activity as it caused 100% mortality by

its rhizome extract at 500 ppm following 48 hours of exposure. Anopheles tessellatus

larvae were subjected to histological analysis after 48 hours of exposure to 300 ppm of

the extract. The histology revealed a disruption in the cuticle and mid-gut epithelium

of the larvae. Additionally, a DNA fragmentation analysis was also performed to detect

possible induction of apoptosis and the results indicated no significant DNA

degradation. The results of this study suggest a potential utilization of the rhizome

extract of Curcuma longa for the control of Anopheles tessellatus larvae. Keywords:

Anopheles tessellatus, Larvae, Curcuma longa, Rhizome, Ethanol

INTRODUCTION

Prevalence of vector-borne diseases


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Vector-borne diseases are one of the major

causes of morbidity and mortality in many

developing countries. Over 350 mosquito

species are vectors of pathogens that cause

life-threatening diseases including malaria,

Japanese encephalitis, lymphatic filariasis,

yellow fever and dengue (Mohankumar,

Shivanna & Achuttan, 2016; Bekele et al.,

2014).

Malaria is caused by Plasmodium sp.

parasites that are transmitted to humans

through the bites of infected female

Anopheline mosquitoes (World Health

Organization, 2017). Anopheles

culicifacies is regarded as the main vector

of malaria in Sri Lanka. In addition to that,

other species that have consistently been

incriminated as malarial vectors are

Anopheles subpictus, Anopheles annularis,

Anopheles varuna, and Anopheles

tessellatus (Gunathilaka et al., 2015).

The global burden of Malaria

According to the latest estimates of World

Health Organization (WHO), there were

216 million cases of malaria and 445 000

deaths reported in 2016 (Figure 1) (World

Health Organization, 2017). Malaria was

endemic in Sri Lanka for centuries and was

a significant public health issue associated

with great economic loss and social

disruption. However, Sri Lanka has

achieved a remarkable success by

eliminating malaria where the last

indigenous case was reported in October

2012 (Karunaweera, Galappaththy &

Wirth, 2014). Although indigenous

transmission of malaria has been

controlled, still there is a risk for the

occurrence of imported malaria cases

mainly from India and African countries.

Therefore, reducing the risk of imported

malaria as well as controlling the vector is

important to maintain the achieved success

(Sri Lanka. Ministry of Health, 2016).

Different approaches have been developed

to lessen the global prevalence of malaria.

Among these strategies, the use of indoor

residual spraying (IRS) and long-lasting

insecticidal nets (LLINs) which are mainly

directed against the adult mosquito have

contributed to a significant reduction of

malaria incidences over the past decade

(World Health Organization, 2017; Samuel

et al., 2016).


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Figure 1. Global malaria incidence rates, by country, 2000-2015 (World Health Organization,

2017)

Conversely, control measures that target

the immature stages of mosquitoes such as

the larval stage, are useful for the control of

malaria in regions where the breeding sites

of mosquitoes are reachable and restricted

in number and size. In contrast to the highly

mobile adult mosquitoes, the immature

stages such as larvae are limited within

comparatively small aquatic habitats and

incapable of escaping the control methods.

Therefore, larval source management

(LSM) plays a progressively significant

role in vector control (Tomass et al., 2011).

Larval and pupal stages of mosquito can be

controlled with the application of synthetic

insecticidal chemicals such as fenthion,

temephos, malathion, diflubenzuron, and

methoprene. However, similar to the adult

vector, these life stages of anopheline

mosquitoes also have been reported to

develop resistance against synthetic

insecticides (Liu et al., 2014; Tomass et al.,

2011).

Phytochemicals as natural insecticides

The use of synthetic insecticides in the

aquatic sources causes many risks to the

humans and environment. In present,

application of many of the synthetic

insecticides in mosquito control programs

has been restricted due to many reasons

including the concern for environmental

sustainability, risks on human health, and

their non-biodegradable nature (Ghosh,

Chowdhury & Chandra, 2012).

Natural products from botanical sources

with insecticidal properties have been

studied in the recent past for the control of

diverse vectors (Parte et al., 2015). Many

researchers have stated the effectiveness of

plant extracts against mosquito larvae

(Kumar et al., 2012). Phytochemicals


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derived from plant sources have been

reported as potential repellents, larvicides,

oviposition attractants, and insect growth

regulators. These phytochemicals can play

a significant role in mosquito control and

thus can interrupt the transmission of

mosquito-borne diseases at both individual

and community level (Srinivasan et al.,

2014).

The effectiveness of phytochemicals

against mosquito larvae may present

significant variations depending on the

species of plant, plant parts used, maturity

of the plant, as well as upon the presented

vector species (Ghosh, Chowdhury &

Chandra, 2012). Hence, there is a necessity

to identify target-specific, easy to use, eco-

friendly and low-cost alternative

insecticidal constituents from plant

resources. Many previous studies have

discovered the presence of insecticidal

plants belonging to diverse families with a

wide geographical distribution across the

world. The crude solvent extracts of many

plant materials have been identified to have

different levels of bioactivity against the

larval stages of mosquitoes (Tomass et al.,

2011).

Biological properties of Curcuma longa

Curcuma longa plant (common name,

turmeric) (Figure 2) is a perennial herb

belonging to the ginger family which is

extensively cultivated in the south and

southeast tropical Asia (Himesh et al.,

2010). The rhizome of turmeric is highly

beneficial and widely been used for

medicinal and culinary purposes. The most

active constituent of Curcuma longa is

curcumin, which is about 2 to 5% of the

species. Turmeric consists of an extensive

variety of phytochemicals including

curcumin, demethoxycurcumin,

bisdemethoxycuicumin, curcumenol,

curcumol, zingiberene, eugenol,

triethylcurcumin, tetrahydrocurcumin,

turmerin, turmeronols and turmerones. The

characteristic yellow color of Curcuma

longa is due to the presence of curcumin,

which comprises curcumin I (94%),

curcumin II (6%) and curcumin III (0.3%).

It has a hydrophobic nature and freely

soluble in ethanol, acetic acid, chloroform

and ketone (Himesh et al., 2010).

Figure 2. The Curcuma longa plant

For the past few decades, extensive

work has been undertaken to evaluate

the pharmacological activities and

biological actions of Curcuma longa

and its extracts. Curcumin

(diferuloylmethane), the chief bioactive

constituent of the plant has been found

to have a broad-spectrum of biological

actions. These include its antioxidant,

anti-carcinogenic, anti-inflammatory,

anti-mutagenic, anticoagulant, anti-

diabetic, antifertility, antibacterial,

antiviral, antifungal, anti-fibrotic, anti-

venom, antiulcer, hypotensive and


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hypocholesteremic properties (Krup,

Prakash & Harini, 2013; Prashar et al.,

2011; Chattopadhyay et al., 2004).

In view of a cumulative interest in

developing insecticides from plant

origins as an alternative to chemical

insecticide, the current study was

performed to evaluate the larvicidal

potential of the ethanolic ribosome

extracts of Curcuma longa against the

Anopheles tessellatus larvae.

METHODOLOGY

Plant material

The fresh rhizomes of Curcuma longa

were collected from a field in

Neelanmahara area of Maharagama, Sri

Lanka (annual temperature of 320C and

rainfall of 4 mm) (Figure 3). The

rhizomes were thoroughly washed

under running tap water, sliced into

smaller pieces and dried in a hot-air

oven at 37oC for 3 days.

Figure 3. The map of Sri Lanka showing

Neelanmahara area (latitude 6.8376267,

longitude 79.9263141 and 25 m above sea

level)

Rearing mosquito larvae

The eggs of Anopheles tessellatus were

allowed to hatch into larvae and procured

from Department of Parasitology, Faculty

of Medicine at the University of Colombo.

Three larvae (n=3) were allocated into

containers, each containing 10.00ml of de-

chlorinated water. Physiological

parameters of the de-chlorinated water

including temperature and pH were

measured. Larval forms were fed on an

appropriate food mixture. Containers were

covered by using nylon net with small pore

size and allowed to adapt to the

environmental conditions for 24hrs.

Larval identification

Stage II and III larvae were placed

individually in petri dishes and observed

under an inverted light microscope (TCM

400) with an objective (x10 and x40).

Using standard larval keys developed for

Sri Lankan anopheline (Amarasinghe,

1992), they were identified to the species

level. Further, larval species identification

was reconfirmed through adult

identification.

Adult mosquito identification

The adult mosquito immersed in 70%

ethanol and refrigerated overnight.

Thereafter, the mosquito was dissected and

parts were mounted by using Canada

balsam. The prepared permanent slides

were observed and identified under the

light microscope (LABOMED) using

standard adult mosquito keys developed for


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Sri Lankan anopheline (Gunathilaka,

2017).

Preparation of plant extract

The dried turmeric was ground to fine

powder form with an electric grinder and

stored at 4oC in air-tight plastic bags

covered with aluminum foils until use

(Figure 4). The ethanol extract of Curcuma

longa was prepared by dissolving 50.0g of

dried powder in 100.0ml of absolute

alcohol. The mixture was left in the dark at

room temperature for 24 hours and shaken

at regular intervals. Thereafter, the mixture

was subjected to stirring at 500 rpm for 20

minutes in a magnetic stirrer. The solution

was filtered using a glass funnel and

Whatman number 1 filter papers. Filtrates

were dried in the fume hood. The

evaporated plant extract was collected into

air-tight containers, covered with

aluminum foils and refrigerated at 4oC until

further use.

Figure 4. The rhizomes (left) and the power

(right) of turmeric

Preparation of stock solution

A 5.00 mg/ml of stock solution was

prepared by dissolving 0.05g of plant

extract in 100.00µl of DMSO. Once the

residue was completely dissolved, it was

further diluted by 9.90ml of de-chlorinated

water (The ultimate concentration of

DMSO in the stock solution was 0.1%).

The stock solution was vigorously vortexed

to obtain a homogenized mixture.

Larvicidal Bioassay test

Three set of second instar larvae triplicates

(n=3) with more or less similar body length

were sorted out (Figure 5) and treated with

different concentration of plant extract

(Table 3). The larvicidal activity of the

turmeric extract was assessed by the WHO

standard larvicidal bioassay test (WHO,

2005). The control set was prepared by

adding 0.1% DMSO. The larval behavior at

regular time intervals and the mortality

were recorded following 2, 4, 24 and 48

hours of exposure. Larvae were kept in a

cool and low light area in the lab for 48

hours in the unfed state. The containers

were covered with the net in order to reduce

evaporation and to facilitate dissolve

oxygen level.

Table 3. The different concentrations of

turmeric extract

Group Treatment

N/C 0.1% of DMSO

C1 25 ppm

C2 50 ppm

C3 100 ppm

C4 300 ppm

C5 500 ppm


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Figure 5. The bioassay setup before (left)

and after (right) the exposure

Histological Procedure

For histological studies, the live C4 larvae

(300ppm) after 48 hours exposure to the

plant extract were fixed in 5 ml of 10%

neutral buffered formalin and refrigerated.

After dehydration in a graded ethanol

series, the larvae were subjected to two

washes in xylene. Thereafter, larvae were

subjected to two paraffin wax baths at 58-

60oC before infiltration by using a wax

dispenser (TPC 2000). Once solidified,

paraffin blocks were kept at 4oC until use.

The paraffin block was sectioned using

rotary microtome (SHANDON FINESSE

325) at 5-7 µm. The sections were mounted

onto glass slides which were coated with

egg albumin. The slides were placed on the

slide dryer (SD 2800) at 38oC for 30

minutes. The slides were observed under

the light microscope (LABOMED) at 100x

to ensure the presence of larval tissue.

The larval tissues were stained using

Hematoxylin and Eosin staining procedure

(Appendix 1) and observed under the light

microscope at 200x to compare the

histological changes with the control

larvae.

DNA fragmentation analysis:

The C2 (100ppm) larvae obtained 24 hours

post-exposure was fixed in 5 ml of ice-cold

PBS. For the DNA extraction procedure,

PBS buffer was removed from the micro-

centrifuge tube. Thereafter, the larvae were

homogenized in Liffton’s buffer and kept

for 30 minutes. Subsequently, Proteinase K

was added to the homogenized sample and

vortexed. The mixture was incubated for 1

hour at 55°C.

This was followed by the addition of

phenol: chloroform: isoamyl alcohol to the

sample. The sample was vortexed and

centrifuged at 12000 rpm for 15 minutes.

The aqueous layer of the sample was

transferred to a new micro-centrifuge tube

and an equal volume of ice-cold

Isopropanol was added. The extract was

stored in -20°C for 30 minutes. Thereafter,

the extract was centrifuged at 12000 rpm

for 15 minutes. The supernatant was

removed from the micro-centrifuge tube

and the pellet was dried. Subsequently,

nuclease-free water was added to micro-

centrifuge tube. The extracted sample was

run on 1% agarose gel at 50V for 10

minutes and at 100V for 1 hour.

A 1.0% agarose gel was placed on the

electrophoresis chamber in a horizontal

position and TBE buffer was added to the

chamber until it covers the gel. Then 8.0µL

ethidium bromide was added to the


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chamber. Subsequently, 10 µL of DNA

extract was mixed with 5µL of gel loading

buffer and loaded into the wells. The lid of

the chamber was closed and the gel was

allowed to run at 100V for 1 hour. Then the

gel was placed in the UV Trans-illuminator

and bands were observed using the

software and the monitor.

Statistical analysis

Statistical analysis was performed using

SPSS version 21.0 statistical software and

Microsoft Excel 2013. Results were

evaluated for its normality. Two-way

ANOVA was performed for normally

distributed data with the significance of

p<0.05. The significant differences further

estimated using Tukey’s pairwise test. The

mean mortality percentages of larvae were

subjected to probit analysis in order to

calculate LC50 values following 24 and 48

hours of exposure along with the regression

equation.

DATA ANALYSIS

Species identification

Larval identification

Anopheline larvae can be distinguished in

the field based on their resting positions in

the water. Due to the absence of siphon, the

larvae belonging to the genus Anopheles

generally position parallel to the water

surface (Figure 6) (Centre of Disease

Control and Prevention, 2017).

Figure 6. Light microscope view of the

Anopheles 4th instar larvae (100x)

Three distinct body regions; head (antennal

hairs, inner clypeal hairs, outer clypeal

hairs, frontal hairs and sutural hairs), thorax

(thoracic palmate hairs, shoulder hairs, pro,

meso and meta thoracic hairs), abdomen

(abdominal tergal plates, palmate hairs in

abdominal segments, lateral hairs in the

abdominal segments) were noted.


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Figure 7: The morphological view of head and the thorax of Anopheles larvae (400x) (A) Seta

1-A simple; 2-C inserted at least as far apart as the distance between 2-C and 3-C on one side

(B) Long thoracic pleural setae 9, 10, 12-P, 9, 10-M and 9, 10-T simple (C) Seta 1-P weak, 2-

5 branched; 1, 2-P arising from separate basal tubercles, only tubercle of 2-P prominent and

sclerotized

Based on the morphological characteristics

of the dorsal view of head (Figure 7A), the

subgenus Cellia was distinguished from the

subgenus Anopheles (Amarasinghe, 1992).

The Sri Lankan anophelines of subgenus

Cellia are categorized into four taxonomic

series: Pyretophorous, Myzomyia,

Neomyzomyia and Neocellia series. The

observed ventral view of thorax (Figure

7B) confirmed the taxonomic series as

Neomyzomyia. Two species, Anopheles

tessellatus, and Anopheles elegans have

been reported under Neomyzomyia series in

Sri Lanka. Based on the features observed

in the dorsal view of prothorax (Figure 7C),

the species was distinguished as Anopheles

Tessellatus from Anopheles elegans

(Gunathilaka, 2017). The species

identification was further confirmed by

observing the morphological features of the

adult mosquito.


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Anopheline adult mosquito identification

Figure 8. Light microscopic view of adult Anopheline mosquito (400X) (A) Wing with 4 or more

darks marks involving both costa and veins R-R1 accessory sector pale (ASP) spot present on

costa and/or subcostal, (B) Femur and tibia speckled (C) The head of the mosquito with a part

of proboscis (D) Apical half of the proboscis pale scaled

The observed morphological features of the

wing of Anopheline (Figure 8A)

distinguished the subgenus Cellia from the

subgenus Anopheles. Among the species

belongs to subgenus Cellia, A. elegans and

A. tessellatus, which belong to

Neomyzomyia series and A. Maculatus and

jamesii group which belong to Neocellia

series feature speckled femur and tibia

(Figure 8B). Based on the characteristics

observed on the proboscis (Figure 8D) the

species was confirmed as Anopheles

tessellatus (Gunathilaka, 2017).

Larvicidal bioassay

Physical characteristics of water used for

the study, such as median temperature 28 ±

0.20C and pH 6.6 ± 0.2 were within the

permissible limits throughout the study

period. The results of Anopheles tessellatus

larval mortality tested against Curcuma

longa crude ethanolic extract are presented

in Table 4. The results of the present study

revealed the high larvicidal activity of

turmeric rhizome extract against Anopheles

tessellatus.


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Table 4. Mortality rates of Anopheles tessellatus larvae following exposure to ethanolic extract

of Curcuma longa

Sample Concentration of the

extract (ppm)

Mortality Rate (%)

24hrs 48hrs

N/C 0 00.00 ± 00.00 00.00 ± 00.00

C1 25 00.00 ± 00.00 00.00 ± 00.00

C2 50 00.00 ± 00.00 11.11 ± 11.11

C3 100 11.11 ± 11.11 33.33 ± 00.00

C4 300 33.33 ± 19.25 66.67 ± 19.25

C5 500 88.89 ± 11.11 100 ± 00.00

An 88.89% and 100% of larvae mortality were observed at the highest dose rate of 500ppm

following 24 and 48 hours of exposure (Figure 9). Further, the effect of larval mortality was

dependent on the concentration of rhizome extract and the exposure time duration.

Figure 9. Percentage of larvae mortality at different hours following treatment with different

concentrations of ethanolic extract of turmeric.


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The highest rate of mortality in larvae was

observed where the highest concentration

of extract (500 ppm) was exposed.

Significant differences were observed

between the mortality rates caused by

different concentrations of the plant extract

against Anopheles tessellatus larvae of

laboratory strains (MS = 8401.388, df = 5,

P = 0.000) (Table 5). In addition to that, the

time or exposure period was also found to

have a statistically significant effect on the

mortality rates (MS = 1512.173, df = 1, P =

0.028). Therefore, P< 0.05, Tukey’s

pairwise tests were performed following

one way ANOVA. The highest

concentration (500 ppm) was found to have

a significantly difference from other tested

concentrations (N/C, P = 0.000; 25 ppm, P

= 0.000; 50 ppm, P = 0.000; 100 ppm, P =

0.000, 300 ppm = P = 0.001).

Table 5. Two-way ANOVA measuring the effects of different concentrations of Curcuma longa

extract on Anopheles tessellatus larvae mortality after 24 h and 48 h of exposure

Source Type III Sum of

Squares

df Mean Square F Sig.

Concentration 42006.938 5 8401.388 30.245 .000

Time 1512.173 1 1512.173 5.444 .028

Concentration * Time 1265.383 5 253.077 .911 .490

Error 6666.667 24 277.778

Total 81110.889 36

Corrected Total 51451.161 35

The result of log-probit analysis (at 95% confidence level) revealed a

gradual decrease in the LC50 value with the increasing exposure period. The

rhizome extract of Curcuma longa presented a high larvicidal activity with LC50 values of 467.74 ppm and 151.36

ppm after 24 and 48 hours of exposure respectively. The concentration-

dependent mortality was detected to be clear, as the mortality rate was

positively correlated with the concentration. However, the low

amount of data and the variance may have an impact on the observed regression coefficient value (Table 6).


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Table 6. Probit analysis of larvicidal efficacy of turmeric rhizome extracts against Anopheles

tessellatus

LC50 (ppm) Regression equation R squared value

24 hours 467.74 y = 2.4399x - 1.5209 0.635302178

48 hours 151.36 y = 2.8696x - 1.2699 0.763292029

Significant behavioral changes were

observed in mosquito larvae exposed to

turmeric extract within 30 minutes of

exposure. The most apparent sign of

behavioral changes observed following 24

hours of exposure in Anopheles tessellatus

larvae was the inability to come on the

surface. The larvae also presented

restlessness, loss of equilibrium and

eventually death. Histological experiments

Histology of Anopheles tessellatus larvae

in the control group presented the normal

form of the body regions. The cuticle layer

of the body was intact with well-developed

mid-gut epithelium exhibiting the brush

border (Figure 10A).

Figure 10. Histological analysis A- Histology of A. tessellatus larvae of control group showing

intact cutical layer of the body (indicated by arrows), B- Histology of the A. tessellatus larvae

treated with 300ppm of turmeric extract for 48 hours showing the disrupted cuticle layer

(indicated by arrows).

After 48 h exposure to Curcuma longa

extract, the cuticle was observed to be

disrupted with partially degraded epithelial

cells began through the dilated basal

membrane (Figure 10 B).

DNA fragmentation analysis


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To examine the effects of the plant extract

on the larval DNA, the larvae which were

exposed to 100ppm of turmeric extract

were analyzed to detect intact DNA on 1%

agarose gel electrophoresis. No apparent

deviations of the larval DNA were detected

on the gel image suggesting that the

exposure to the plant extract does not affect

the larval structure in molecular level

(Figure 11).

Figure 11. The gel image of DNA analysis.

Lane M: molecular ladder; Lane 1:

negative control; Lane 2: DNA sample of

the exposed larva analysis. Lane M:

molecular ladder; Lane 1: negative

control; Lane 2: DNA sample of the

exposed larva

DISCUSSION

Vector control is crucial to prevent the

proliferation of mosquito-borne diseases

and to enhance the quality of public health

(Ghosh, Chowdhury & Chandra, 2012).

The extensive usage of synthetic organic

insecticides in mosquito control operation

during the last five decades has caused the

development of physiological resistance in

target vector population and accumulation

of toxic substances in the environment.

This has necessitated the need of an

alternative which is bio-degradable,

environmentally safe, and easy to use as a

vector control measure (Samuel et al.,

2016; Kumar et al., 2012).

Numerous researchers in the field of vector

control have discovered the effectiveness

of different phytochemicals of botanical

origin against different mosquito species

(Rawani et al., 2017). Among these

botanical sources, turmeric has been long

found to exhibit numerous properties along

with potential larvicidal activity (Krup,

Prakash & Harini, 2013; Prashar et al.,

2011; Chattopadhyay et al., 2004).

The 24 hours bioassay is a major tool which

is applied to evaluate the toxicity of

phytotoxins, and many researchers have

been using this method to measure the toxic

effects of different plant extractions against

mosquitoes (Viji & Nethaji, 2015). The

current study discovered the toxic nature of

ethanolic rhizome extract of Curcuma

longa that demonstrated its larvicidal

activity against the malaria vector,

Anopheles tessellatus. The mortality

percentage of larvae was detected to be

increased with the increasing concentration

of extract and the exposure duration. The

result of log-probit analysis (at 95%

confidence level) revealed a significant

reduction in LC50 value following 48 hours

of exposure (LC50 = 151.36 ppm) in

comparison to LC50 value at 24 hours of

exposure (LC50 = 467.74 ppm). This may

suggest that the efficacy of the plant

compounds which are responsible for the

larval mortality readily increases with the

increasing time of exposure. Conversely, a

reduction in LC50 values with the

increasing exposure period was also

reported in case of aqueous extract of

1000 bp


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Curcuma longa tested against Anopheles

stephensi. However, the reported activity is

more effective compared to the activity in

the present report (Singha & Chandra,

2011).

The ethanolic extract of Curcuma longa is

reported to contain 22.6% curcumin, 6.1%

desmethoxycurcumin, and 15.4% ar-

turmerone with 6.6%

bisdesmethoxycurcumin, which may

responsible for its larvicidal activity (Ali,

Wang & Khan, 2015). In contrast to the

crude extract, the essential oil of Curcuma

longa has been reported to possess much

higher potential as a natural larvicide.

Studies suggest that the larvicidal effects of

essential oil of Curcuma longa may be due

to the greater amount of Phytol (18.89%)

(Albert et al., 2014). Curcumin is presently

been used in many clinical trials and

interestingly, it is found to be non-toxic to

humans. The non-toxic nature makes it a

very attractive applicant for consequent

field experiments with expected

insignificant non-specific species toxicity

and environmental inferences (Sagnoua et

al., 2012).

Other members of the genus Curcuma have

also been reported for their larvicidal

activity towards mosquito larvae. For

instance, Sukari et al., (2010) have

observed the larvicidal action Curcuma

heyneana, Curcuma xanthorrhiza , and

Curcuma mangga, against the dengue

mosquito larvae of Aedes aegpyti. The

authors further reported that a high toxicity

against larvae of Aedes aegypti was

observed in the cases of hexane extract of

C. xanthorrhiza with a LC50 value of 26.4

µg/ml, and the petroleum ether extract of C.

heyneana with a LC50 value of 34.9 µg/ml.

Correspondingly, The LC50 value of

Curcuma aromatica on Japanese

encephalitis vector Culex vishnui larvae

was reported to be 17.25 ppm following 72

hours of exposure (Mallick, Bhattacharya

& Chandra, 2014).

In this study, the observed behavioral

changes of mosquito larvae following

exposure to plant extract were compatible

with other studies. Viji and Nethaji (2015)

reported similar behavioral effects on

larvae of A. aegpyti following exposure to

crude ethanolic extract of C. longa.

Conversely, Remia and Logaswamy (2010)

also reported these behavioral effects in the

cases of Catharanthus roseus and Lantana

camara extracts where the effect of

Catharanthus roseus was found to be more

pronounced after exposure. The authors

further stated that these effects may be due

the presence of neurotoxic compounds in

the plant materials.

Besides causing death to mosquito larvae,

the effect of intoxication also manifests

through the aberration of structures. It is

evident as Aedes aegypti larvae treated with

extracts of natural compounds such as dried

fruits of peppercorns and red seaweed

Laurencia dendroidea has been reported to

have darkening and shrunken cuticle of

anal papillae after the exposure (Bianco et

al., 2013; Kumar, Warikoo & Wahab,

2010). In this study, the observed

histological variations of Anopheles

tessellatus following the exposure to

turmeric extract presented high structural

impairments including the cuticle and the

mid-gut.

As the main absorption area in mosquito

gut, midgut owns fine microvilli in the apex

of cells (Alves, Serrão & Melo, 2010). In

the present study, histological variations

were observed in the mid-gut of Anopheles

tessellatus including partial degradation of

the epithelial cells and disruption of the


16

microvilli and the basal membrane with the

loss their normal appearance. Al-Mehmadi

& Al-Khalaf (2010) have reported that such

impairments may lead to larval mortality

due to the interaction of the gut contents

with the hemolymph. These alternations

were in covenant with previous studies.

The mid-gut of Culex quinquefasciatus

larvae that exposed to Murraya paniculata

leaf extract was presented with separated

epithelial cells from the basal membrane,

separated elongations protruded into the

lumen, and disrupted the appearance of

microvilli (Kjanijou et al., 2012). Similar

features including epithelial cell separation

from the basal membrane have been

observed in Culex quinquefasciatus

following exposure to Melia azedarach

extract (Al-Mehmadi & Al-Khalaf, 2010).

Active compounds of Curcuma longa may

cause disruption of the midgut epithelium,

where its constituents are been absorbed.

Irrespective of the type of constituents

used, the correspondence of detrimental

changes observed in the different species

indicates that these variations are a mutual

response to cellular intoxication (Kjanijou

et al., 2012).

Larvicidal substances are also been

reported to cause protein and DNA

destruction where a reduction of total body

protein and DNA content were observed in

the red cotton bug Dysdercus cingulatus

larvae treated with P. pavonica extract

(Sahayaraj & Kalidas, 2011). Such

reduction can result in prolongation of

larval duration and reduction of adult

emergence (Yu et al., 2015). Moreover, a

study by Magalhães et al., (2009) on

Schistosoma mansoni have reported that

the increasing concentration curcumin can

lead to an increased larval mortality,

decrease worm viability and reduced egg

production which may associate with

transcriptional repression (Morais et al.,

2013). However, the observed result in

DNA fragmentation analysis in the current

study did not present any significant

changes in larval DNA after exposure to

Curcuma longa extract.

The present study evaluated the efficacy of

ethanolic extract of rhizome of Curcuma

longa against the second instar larvae of

Anopheles tessellatus. Although apparent

histological alternations were observed in

larvae following exposure to Curcuma

longa, no significant DNA degradations

were observed at the molecular level. Thus,

during the present study, the observed

disruption of the cuticle and the mid-gut of

the larvae as well as the behavioral changes

including reduced mobility, may be

accountable for larval mortality in this

bioassay experiment.

FUTURE WORK

The results of this study which revealed the

efficacy of Curcuma longa ethanolic

extract as a potential larvicidal agent

against Anopheles tessellatus can be

decoded to affect diverse aspects of

mosquito control agent development. As

curcumin is the major bioactive compound

in turmeric which has been found to be

responsible for it larvicidal activity,

isolation of curcumin from C. longa may

offer a high efficacy against the target

vector population.

Preparation of solvent extracts initially

from non-polar to polar chemicals is

essential to determine the most effective

solvent for the extraction process.

Moreover, the application of evaporation

methods with increase precision in order to

obtain the solid residue from the liquid

solvent may lead to the determination of


17

lethal dose with a higher accuracy. The

column chromatography and thin layer

chromatography can be used to purify the

phytochemicals of turmeric and thereby

analyze its larvicidal potentiality.

Furthermore, a field evaluation of its

larvicidal activity can be performed against

other instar stages of Anopheline larvae as

well as on different Anopheles species.

ACKNOWLEDGEMENT

I am indebted to my college, BMS, School

of Science, the University of Northumbria

for the great opportunity they gave me to

pursue this study. I would like to express

my sincere gratitude to my supervisor, Ms.

Koshala De Silva, for her immense

guidance, advice, support, constant

encouragement and constructive comments

throughout this project. I am also obliged to

Dr. Sajani Diaz, the Dean, and Dr, Mathi

Kandiah, the Deputy Dean of BMS for their

kind support and permission to conduct this

study.

REFERENCES

Albert O.O., Syed, A.M., Yogananth, N.,

Anuradha, V., Ferosekhan, M. &

Munees, P.M. (2014), “Safety and

efficacy of essential oil from Curcuma

longa against Aedes aegypti and

Anopheles stephensi mosquito

vectors”, International Journal of

Comprehensive Research in

Biological Sciences, Vol. 1, No. 1, pp.

36-43.

Ali, A., Wang, Y. & Khan, I.A. (2015),

“Larvicidal and biting deterrent

activity of essential oils of Curcuma

longa, Ar-turmerone, and

Curcuminoids against Aedes aegypti

and Anopheles quadrimaculatus

(Culicidae: Diptera)”, Journal of

Medical Entomology, Vol. 52, No. 5,

pp. 979-986.

Al-Mehmadi, R.M. & Al-Khalaf, A.A. (2010),

“Larvicidal and histological effects of

Melia azedarach extract on Culex

quinquefasciatus Say larvae (Diptera:

Culicidae)”, Journal of King Saud

University – Science, Vol. 22, No. 2,

pp.77-85.

Alves, S.N., Serrão , J.E. & Melo A.L. (2010),

“Alterations in the fat body and midgut

of Culex quinquefasciatus larvae

following exposure to different

insecticides”, Micron, Vol. 41, pp.

592-597.

Amarasinghe, F.P. (1992), “A guide to the

identification of the Anopheline

mosquitoes (Diptheria:Culicidae) of

Sri Lanka”, Ceylon Journal of Science,

Vol. 22, pp: 1-13

Bekele, D., Petros, B., Tekie, H. & Asfaw, Z.

(2014), “Larvicidal and adulticidal

effects of extracts from some

indigenous plants against the malaria

vector, Anopheles Arabiensis

(Diptera: Culicidae) in Ethiopia”,

Journal of Biofertilizers &

Biopesticides, Vol. 5, No. 2, pp. 144

Bianco, E.M., Pires, L., Santos, G.K., Dutra,

K.A., Reis, T.N., Vasconcelos, E.R.,

Cocentinoc, A.L.M. & Navarrob,

D.M.A.F. (2013), “Larvicidal activity

of seaweeds from northeastern Brazil

and of a halogenated sesquiterpene

against the dengue mosquito Aedes


18

aegypti”, Industrial Crops and

Products, Vol. 43, pp. 270–275.

Centre of Disease Control and Prevention

(2017), Anopheles Mosquitoes

[online]

https://www.cdc.gov/malaria/about/bi

ology/mosquitoes/ visited 20 April

2017.

Chattopadhyay, I., Biswas, K.,

Bandyopadhyay, U. & Banerjee, R.K.

(2004), “Turmeric and curcumin:

Biological actions and medicinal

applications”, Current science, Vol.

87, No. 1, pp. 44-53.

Ghosh, A., Chowdhury, N., & Chandra, G.

(2012), “Plant extracts as potential

mosquito larvicides”, The Indian

Journal of Medical Research, Vol.

135, No.5, pp. 581–598.

Gunathilaka, N. (2016), “Illustrated key to the

adult female Anopheles (Diptera:

Culicidae) mosquitoes of Sri Lanka”,

Applied Entomology and Zoology,

Vol. 52, pp: 69-77.

Gunathilaka, N., Hapugoda, M.,

Abeyewickreme, W. &

Wickremasinghe, R. (2015),

“Entomological investigations on

malaria vectors in some war-torn

Areas in the Trincomalee district of Sri

Lanka after settlement of 30-year civil

disturbance”, Malaria Research and

Treatment, Vol. 2015, pp. 1-11

Himesh, S., Patel, S.S., Govind, N. & Ak, S.

(2010), “Qualitative and quantitative

profile of curcumin from ethanolic

extract of Curcuma longa”,

International Research Journal of

Pharmacy, Vol. 2, No. 4, pp. 180-184.

Karunaweera, N.D., Galappaththy, G.N.L. &

Wirth, D.F. (2014), “On the road to

eliminate malaria in Sri Lanka: lessons

from history, challenges, gaps in

knowledge and research needs”,

Malaria Journal, Vol. 13, pp. 59

Kjanijou, M., Jiraungkoorskul, K., Kosai, P. &

Jiraungkoorskul, W. (2012), “Effect of

Murraya paniculata leaf extract against

Culex quinquefasciatus larva”, Asian

Journal of Biological Sciences, Vol. 5,

pp. 201-208.

Krup, V., Prakash, L.H. & Harini, A. (2013),

“Pharmacological activities of

turmeric (Curcuma longa linn): A

review”, Journal of Homeopathic and

Ayurvedic Medicine, Vol. 2, pp. 133.

Kumar, S., Wahab, N., Mishra, M., & Warikoo,

R. (2012), “Evaluation of 15 local

plant species as larvicidal agents

against an Indian strain of dengue

fever mosquito, Aedes aegypti L.

(Diptera: Culicidae)”, Frontiers in

Physiology, Vol. 3, pp. 104.

Kumar, S., Warikoo, R. & Wahab, N. (2010),

“Larvicidal potential of ethanolic

extracts of dried fruits of three species

of peppercorns against different instars

of an Indian strain of dengue fever

mosquito, Aedes aegypti L. (Diptera:

Culicidae)”, Parasitology Research,

Vol. 107, No. 4, pp. 901-907.

Liu, X. C., Liu, Q., Zhou, L. & Liu, Z.L. (2014),

“Evaluation of larvicidal activity of

the essential oil of Allium

macrostemon Bunge and its selected

https://www.cdc.gov/malaria/about/biology/mosquitoes/

https://www.cdc.gov/malaria/about/biology/mosquitoes/


19

major constituent compounds against

Aedes albopictus (Diptera:

Culicidae)”, Parasites & Vectors, Vol.

7, pp. 184

Magalhães L.G., Machado, C.B., Morais, E.R.,

De Carvalho, M.É.B., Soares, C.S, Da

Silva, S.H. & Rodrigues, V. (2009),

“In vitro schistosomicidal activity of

curcumin against Schistosoma

mansoni adult worms”, Parasitology

Research, Vol. 104, No. 5, pp. 1197–

1201.

Mallick, S., Bhattacharya, K. & Chandra, G.

(2014), “Mosquito larvicidal

potentiality of wild turmeric, Curcuma

aromatica rhizome, extracts against

Japanese Encephalitis vector Culex

vishnui group”, Journal of Mosquito

Research, Vol. 4, No. 19, pp. 1-6.

Mohankumar, T.K., Shivanna, K.S., &

Achuttan, V.V. (2016), “Screening of

methanolic plant extracts against

larvae of Aedes aegypti and

Anopheles stephensi in Mysore”,

Journal of Arthropod-Borne Diseases,

Vol. 10, No. 3, pp. 303–314.

Morais, E.R., Oliveira, K.C., Magalhães, L.G.,

Moreira, É.B., Verjovski-Almeida, S.

& Rodrigues, V. (2013), “Effects of

curcumin on the parasite Schistosoma

mansoni: A transcriptomic approach”,

Molecular Biochemical Parasitology,

Vol. 187, No. 2, pp. 91–97.

Parte, S.G., Kharat, A.S., Mohekar, A.D.,

Chavan, J.A., Jagtap, A.A., Mohite,

A.K. & Patil, R.N. (2015), “Efficacy

of plant extracts for management of

Cimex lectularius (Bed Bug)”,

International Journal of Pure &

Applied Bioscience, Vol. 3, No. 2, pp.

506-508.

Prashar, D., Khokra, S.L., Purohit, R. &

Sharma, S. (2011), “Curcumin: A

potential bioactive agent”, Research

Journal of Pharmaceutical, Biological

and Chemical Sciences, Vol. 2, No. 4,

pp. 44-52.

Rawani, A., Ray, A.S., Ghosh, A., Sakar, M. &

Chandra, G. (2017), “Larvicidal

activity of phytosteroid compounds

from leaf extract of Solanum nigrum

against Culex vishnui group and

Anopheles subpictus”, BMC Research

Notes, Vol. 10, pp. 135.

Remia, K.M. & Logaswamy, S. (2010),

“Larvicidal efficacy of leaf extract of

two botanicals against the mosquito

vector Aedes aegypti (Diptera:

Culicidae)”, Indian Journal of Natural

Product Resources, Vol. 1, No. 2, pp.

208-212.

Sagnoua, M., Mitsopouloub, K.P.,

Koliopoulosd, G., Pelecanoua, M.,

Couladourosb, E.A. & Michaelakise,

A. (2012), “Evaluation of naturally

occurring curcuminoids and related

compounds against mosquito larvae”,

Acta Tropica, Vol. 123, No. 3, pp. 190-

195.

Sahayaraj, K & Kalidas, S. (2011), “Evaluation

of nymphicidal and ovicidal effect of

seaweed, Padina pavonica (Linn.)

(Phaeophyceae) on cotton pest,

Dysdercus cingulatus (Fab.)”, Indian

Journal of Geo-Marine Sciences, Vol.

40, No. 1, pp.125-129.


20

Samuel, M., Oliver, S.V., Coetzee, M., &

Brooke, B.D. (2016), “The larvicidal

effects of black pepper (Piper nigrum

L.) and piperine against insecticide

resistant and susceptible strains of

Anopheles malaria vector

mosquitoes”, Parasites & Vectors,

Vol. 9, pp. 238.

Singha, S. & Chandra, G. (2011), “Mosquito

larvicidal activity of some common

spices and vegetable waste on Culex

quinquefasciatus and Anopheles

stephensi”, Asian Pacific Journal of

Tropical Medicine, Vol. 4, No.4, pp.

288-293.

Sri Lanka. Ministry of Health (2016), End

Malaria for Good - Can Sri Lanka

Prevent Reintroduction. Weekly

Epidemiological Report [Online]

http://www.epid.gov.lk/web/images/p

df/wer/2016/vol_43_no_14-

english.pdf visited 13 March 2017.

Srinivasan, R., Natarajan, D., Karthi, S. &

Shivakumar, M.S. (2014), “Chemical

composition and larvicidal activity of

Elaeagnus indica Servett.

(Elaeagnaceae) plant leaf extracts

against dengue and malaria vectors”,

International Journal of Mosquito

Research, Vol. 1, No. 4, pp. 66-71.

Sukari, M.A., Rashid, N.Y., Neoh, B.K., Bakar,

N.H.A., Riyanto, S. & Ee, G.C.L.

(2010), “Larvicidal activity of some

Curcuma and Kaempferia rhizome

extracts against dengue fever mosquito

Aedes aegypti Linnaeus (Diptera:

Culicidae)”, Asian Journal of

Chemistry, Vol. 22, No.10, pp. 7915-

7919.

Tomass, Z., Hadis, M., Taye, A., Mekonnen, Y.

& Petros, B. (2011), “Larvicidal

effects of Jatropha curcas L. against

Anopheles arabiensis (Diptera:

Culicidea)”, Momona Ethiopian

Journal of Science, Vol. 3, No.1, pp.

52-64.

Viji, S. & Nethaji, S. (2015), “Larvicidal

efficacy of rhizome extracts of Acorus

calamus & Curcuma longa against the

dengue fever mosquito vector Aedes

aegypti”, International Journal of

Innovative Research in Science,

Engineering and Technology, Vol. 4,

No.1, pp. 11375 – 11379.

World Health Organization (2005), Guidelines

for laboratory and field testing of

mosquito larvicides [Online]

http://apps.who.int/iris /bitstream/1066

5/69101/1/WHO_CDS_WHOPES_G

CDPP_2005.13.pdf visited 5 April

2017

World Health Organization (2017), Fact sheet

about Malaria [online]

http://www.who.int/mediacentre/facts

heets/fs094/en/ visited 16 April 2017.

Yu, K., Wong, C., Ahmad, R. & Jantan, I.

(2015), “Larvicidal activity, inhibition

effect on development,

histopathological alteration and

morphological aberration induced by

seaweed extracts in Aedes aegypti

(Diptera: Culicidae)”, Asian Pacific

Journal of Tropical Medicine, Vol. 8,

No. 12, pp. 1006–1012.

http://www.epid.gov.lk/web/images/pdf/wer/2016/vol_43_no_14-english.pdf



http://www.who.int/mediacentre/factsheets/fs094/en/

http://www.who.int/mediacentre/factsheets/fs094/en/


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APPENDIX 1: Hematoxylin and Eosin

staining procedure (H & E Staining)

The mounted larval tissue slides were

stained with using following H & E

Staining Procedure.

• Xylene I - 5 minutes

• Xylene II - 5 minutes

• 100% Ethanol I - 3 minutes

• 100% Ethanol II - 3 minutes

• 95% Ethanol - 3 minutes

• 70% Ethanol -3 minutes

• Distilled Water - 30 Seconds

• Hematoxylin - 45 Seconds

• Tap water – 2 to 3 minutes

• Distilled water – 1 to 2 minutes

• Acid Alcohol - 15 Seconds

• 70% Ethanol -3 minutes

• Eosin – 5 to10 Seconds

• 95% Ethanol - 30 Seconds

• 100% Ethanol I - 3 minutes

• 100% Ethanol II - 3 minutes

• Xylene I - 3 minutes

• Xylene II -3 minutes

After the staining procedure, a drop of

Canada balsam was put on the section and

placed a cover slip, press to remove air

bubbles. Let it dry for 5-10 minutes.


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LARVICIDAL EFFECTS OF CURCUMA LONGA …multidisciplinaryjournal.globalacademicresearchinstitute...Interntional Conference on Health and Medicine 6 Sri Lankan anopheline (Gunathilaka,